The incidence of fungal infections and, in particular, Candida infections, the fourth most common nosocomial pathogen, has continued to increase over the past 20 years. This increase is due to the increasing numbers of patients subjected to severe immunosuppression as a result of transplant procedures, chemotherapy regimens, advances in medical and surgical therapies, and the increased use of invasive devices such as intravascular central lines. While the majority of invasive fungal disease (IFD) is caused by species of Candida and Aspergillus, other rare fungal pathogens have become more prevalent in recent decades and are associated with exceedingly high mortalities. Due to the slow turnaround time of current diagnostic methods, proper treatment of IFD is often delayed, contributing to the high mortality rates associated with this disease. Microbiologic culture, the current diagnostic standard, requires, on average, 48-72 hours for completion, and fails to detect roughly 50% of fungal species. As proper treatment of IFD calls for prompt and accurate diagnosis, an unmet need in the area of fungal diagnostics is a platform that can provide 1) early detection 2) sensitive results, 3) species level discrimination and 4) multiplex capability. Several molecular approaches have been developed to address this need but have failed to meet all these requirements. Our Reverse Line Blot (RLB) based assay uses PCR followed by hybridization with species specific probes, offering a solution to this unmet need by providing a sensitive and rapid (less than 8 hour) test for several fungal pathogens in a multiplex format. In this Phase I application we propose development of RLB based assays for the detection of 9 species of Candida, 5 species of Aspergillus, and several more rare species of IFD, including H. capsulatum, Fusarium spp, Zygomycetes, Cryptococcus spp, and Coccidioides spp. A major milestone of Phase I will be the development and validation of a multiplexed RLB-based, PCR assay for the detection of the majority of fungal pathogens implicated in IFD. We will validate our RLB assays in clinical blood and BAL samples obtained from several collaborators with expertise in mycology including Randall Hayden of St. Jude Medical Center, Peter Pappas of the University of Alabama, Luis Ostrosky of the University of Texas, David Snydman of Tufts University and Mary Brandt of the Centers for Disease Control. Our end product will be developed further in Phase II, where we will transition our open system to a closed system that performs the PCR and hybridization reactions in a dedicated instrument, designed and tested in collaboration with our engineering partner, Wi Engineering. This product offers a rapid, sensitive and multiplexed approach that meets the requirements of an ideal diagnostic platform for IFD. By significantly reducing time to detection, this innovative platform will lead to changes in the treatment of IFD, most notably, faster treatment with a targeted anti-fungal.